Exonuclease III-assisted gold nanoparticle conjugated DNA walker for sensitive and specific detection of circulating tumor DNA

Circulating tumor DNA (ctDNA), a critical biomarker for cancer diagnostics and therapeutic monitoring, remains challenging to analyse due to its low concentration and short half-life in blood circulation. Conventional methods, such as polymerase chain reaction (PCR) and DNA sequencing, are limited by high costs, laborious workflows, and susceptibility to false positives caused by non-target amplification. To overcome these barriers, we developed an Exonuclease III (Exo III)-assisted DNA walker system integrated with gold nanoparticles (AuNPs), also termed Au-3D walkers, for ultrasensitive ctDNA detection. This platform exploits the sequence-independent activity of Exo III to enable continuous target recycling through a burnt-bridge amplification mechanism. The walker selectively binds to double-stranded DNA (dsDNA) formed between ctDNA and AuNP-bound track strands, triggering Exo III-mediated cleavage of the track strand. This process generates a fluorescence signal while releasing ctDNA for subsequent recognition cycles, achieving sensitive detection without requiring enzyme-specific sequence design. The modular architecture of Au-3D walkers supports autonomous, multi-cycle ctDNA recognition on AuNP surfaces, addressing limitations of immobilized walker systems and enhancing operational efficiency. By integrating the programmability of DNA nanomachines with the specificity of Exo III, this strategy offers a robust, scalable solution for ctDNA analysis. The AuNP-based platform leverages the high surface-to-volume ratio and stability for immobilizing DNA walkers to further amplify signal output and improve detection sensitivity. Furthermore, this design circumvents the need for complex target-activation sequences or enzyme-recognition motifs, broadening its applicability in complex biosensing environments. Experimental validation demonstrates an ultrasensitive detection limit. This work provides a versatile tool for ctDNA-related clinical and pathological research, with significant potential for advancing early cancer diagnostics, monitoring treatment responses, and enabling precision medicine. The proposed strategy bridges the gap between nanotechnology and molecular diagnostics, offering a foundation for future innovations in liquid biopsy technologies.